The Role Of Calcium Ions In The Cellular Entry Of Influenza A Virus

Influenza A virus(IAV)has caused global and regional pandemics for many times,causing great damage to public safety and national economy.The high variability of IAV's HA and NA gene sequences caused difficulties the development of anti-influenza drugs.The comprehensive study of the IAV entry into cells and its key molecular events can provide the theoretical foundation for the development of the anti-influenza drugs and control of influenza virus infection.In this paper,we mainly study the role of extracellular calcium ions(Ca²⁺)in the entry of IAV.Firstly,using the Ca²⁺sensor GCa MP6f to label cytosolic Ca²⁺,we studied the relation between IAV infection and cytosolic Ca²⁺concentration,and found that IAV infection was accompanied with an elevation of cytosolic Ca²⁺concentration.Abolishing the influx of extracellular Ca²⁺totally suppressed the phenomenon of increased cytosolic Ca²⁺concentration during virus infection,however,inhibition of endoplasmic reticulum Ca²⁺release only partially inhibited the increase of Ca²⁺concentration.It suggested that IAV infection was accompanied with cytosolic Ca²⁺concentration increase caused by Ca²⁺influx from the extracellular matrix into cells and Ca²⁺influx-induced endoplasmic reticulum Ca²⁺release into the cytosol.Then,we discussed the role of extracellular Ca²⁺influx in the entry of IAV,and found that abolishing extracellular Ca²⁺influx could inhibit the internalization of IAV in host cells without affecting the attachment of viruses on the cells surface,indicating that extracellular Ca²⁺influx played a key role in the process of IAV entry into host cells through endocytosis.By statically inhibiting different endocytic pathways of IAV and tracking trajectories of IAV entry into cells,we found that Ca²⁺influx was mainly involved in the process of IAV entering cells via clathrin-mediated endocytosis(CME),but not involved in the process of IAV entering cells via clathrin-independent endocytosis.By severally blocking the internalization steps of IAVs'CME,we found that Ca²⁺influx occurred after IAV recruiting dynamin and before IAV transporting along the microtubules to the the cell interior,which implied that extracellular Ca²⁺influx might participate in the dynamin-promoted vesicle maturation and membrane fission in CME of IAVs.In addition,we found that Ca²⁺influx did not participate in the uptake of two nutrients(transferrin and low density lipoprotein)entering cells via CME,suggested that Ca²⁺influx might play some specific roles in the CME of IAVs.These findings contribute to the more comprehensive understanding of the mechanism of IAV entry into host cells and the important inspiration significance of developing the anti-influenza drugs.